Polypeptides having haloperoxidase activity

ABSTRACT

The present invention relates to isolated polypeptides having haloperoxidase activity. The invention also relates to methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims under 35 U.S.C. 119 priority from or the benefitof Danish application PA 2000 00626 filed Apr. 14, 2000 and U.S.application Ser. No. 60/202,249, filed May 5, 2000, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to isolated polypeptides havinghaloperoxidase activity as well as methods for producing and using thepolypeptides.

BACKGROUND

Haloperoxidases are widespread in nature being produced by mammals,plants, algae, lichen, bacteria, and fungi. Haloperoxidases are probablythe enzymes responsible for the formation of naturally occurringhalogenated compounds. There are three types of haloperoxidases,classified according to their specificity for halide ions:Chloroperoxidases (E.C. 1.11.1.10) which catalyze the chlorination,bromination and iodination of compounds; bromoperoxidases which showspecificity for bromide and iodide ions; and iodoperoxidases (E.C.1.11.1.8) which solely catalyze the oxidation of iodide ions.

The first discovered haloperoxidases were determined to contain heme asa prosthetic group or co-factor. However, more recently, it has becomeapparent that there are numerous non-heme haloperoxidases as well.Bacterial haloperoxidases have been found with no prosthetic group. Inaddition, a number of other non-heme haloperoxidases have been shown topossess a vanadium prosthetic group. Haloperoxidases containing avanadium prosthetic group are known to include at least two types offungal chloroperoxidases from Curvularia inaequalis (van Schijndel etal., 1993, Biochimica Biophysica Acta 1161:249-256; Simons et al., 1995,European Journal of Biochemistry 229: 566-574; WO 95/27046) andCurvularia verruculosa (WO 97/04102).

Haloperoxidases, like other oxidoreductases, are of current interestbecause of their broad range of potential industrial uses.

It is an object of the present invention to provide improvedpolypeptides having haloperoxidase activity and nucleic acid encodingthe polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havinghaloperoxidase activity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 80%homology with the amino acid sequence of SEQ ID NO:2;

(b) a polypeptide encoded by a nucleic acid sequence which hybridizesunder medium stringency conditions with (i) the nucleotide sequence ofSEQ ID NO:1, (ii) a subsequence of (i) of at least 100 nucleotides, or(iii) a complementary strand of (i) or

(c) a variant of the polypeptide having an amino acid sequence of SEQ IDNO:2 comprising a substitution, deletion, and/or insertion of one ormore amino acids;

(d) an allelic variant of (a) or (b);

(e) a fragment of (a), (b), or (d) that has haloperoxidase activity; and

(f) a polypeptide having more than 50% residual activity after 15minutes incubation at 70° C. and pH 7.

The present invention also relates to methods for producing and usingthe polypeptides.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having HaloperoxidaseActivity

The term “haloperoxidase activity” as defined herein catalyzes theoxidation of a halide ion (X=Cl—, Br—, or I—) in the presence ofhydrogen peroxide (H₂O₂) to the corresponding hypohalous acid (HOX):

H₂O₂+X−+H+→H₂O+HOX

For purposes of the present invention, haloperoxidase activity isdetermined according to the procedure described in “Haloperoxidaseassays” in the Examples.

In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofhomology to the amino acid sequence of SEQ ID NO:2 of at least about80%, preferably at least about 90%, more preferably at least about 95%,and most preferably at least about 97%, which have haloperoxidaseactivity (hereinafter “homologous polypeptides”). In a preferredembodiment, the homologous polypeptides have an amino acid sequencewhich differs by five amino acids, preferably by four amino acids, morepreferably by three amino acids, even more preferably by two aminoacids, and most preferably by one amino acid from the amino acidsequence of SEQ ID NO:2. For purposes of the present invention, thedegree of homology between two amino acid sequences is determined byusing GAP version 8 from the GCG package (Genetics Computer Group, 575Science Drive, Madison, Wis. 53711, USA) with standard penalties forproteins: GAP weight 3.00, length weight 0.100, Matrix described inGribskov and Burgess, Nucl. Acids Res. 14(16); 6745-6763 (1986).

Preferably, the polypeptides of the present invention comprise the aminoacid sequence of SEQ ID NO:2 or an allelic variant thereof; or afragment thereof that has haloperoxidase activity. In a more preferredembodiment, the polypeptide of the present invention comprises the aminoacid sequence of SEQ ID NO:2. In another preferred embodiment, thepolypeptide of the present invention consists of the amino acid sequenceof SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof thathas haloperoxidase activity. In another preferred embodiment, thepolypeptide of the present invention consists of the amino acid sequenceof SEQ ID NO:2.

A fragment of SEQ ID NO:2 is a polypeptide having one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

In a second embodiment, the present invention relates to isolatedpolypeptides having haloperoxidase activity which are encoded by nucleicacid sequences which hybridize under medium stringency conditions,preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with (i) the nucleotide sequence of SEQ ID NO:1, (ii) asubsequence of (i), or (iii) a complementary strand of (i) or (ii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y. The subsequenceof SEQ ID NO:1 may be at least 100 nucleotides or preferably at least200 nucleotides. Moreover, the subsequence may encode a polypeptidefragment, which has haloperoxidase activity. The polypeptides may alsobe allelic variants or fragments of the polypeptides that havehaloperoxidase activity.

The nucleic acid sequence of SEQ ID NO:1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO:2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having haloperoxidase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA, which hybridizes with the probes describedabove and which encodes a polypeptide having haloperoxidase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1 or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a labeled nucleic acid probe corresponding to the nucleicacid sequence shown in SEQ ID NO:1, its complementary strand, or asubsequence thereof, under low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO:2, or a subsequencethereof. In another preferred embodiment, the nucleic acid probe is SEQID NO:1. In another preferred embodiment, the nucleic acid probe is thenucleic acid sequence contained in the pUC19 derived plasmid containedin Escherichia coli DH10B, deposited as DSM 13442, wherein the nucleicacid sequence encodes a polypeptide having haloperoxidase activity.

For long probes of at least 100 nucleotides in length, low to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and either 25% formamide for low stringencies, 35% formamidefor medium and medium-high stringencies, or 50% formamide for high andvery high stringencies, following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 50° C. (low stringency), more preferablyat least at 55° C. (medium stringency), more preferably at least at 60°C. (medium-high stringency), even more preferably at least at 65° C.(high stringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SSC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third embodiment, the present invention relates to variants of thepolypeptide having an amino acid sequence of SEQ ID NO:2 comprising asubstitution, deletion, and/or insertion of one or more amino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of SEQ ID NO:2 by an insertion or deletion of one ormore amino acid residues and/or the substitution of one or more aminoacid residues by different amino acid residues. Preferably, amino acidchanges are of a minor nature, that is conservative amino acidsubstitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue;

a small linker peptide of up to about 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions, which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

In a fourth embodiment, the present invention relates to isolatedpolypeptides having a residual activity of at least 50% residualactivity, preferably at least 60% residual activity after 15 minutesincubation at 70° C. and pH 7. In a preferred embodiment, thepolypeptides of the invention retain at least 50% residual activity,preferably at least 80% residual activity after 15 minutes incubation at60° C. and pH 7.

In a preferred embodiment, the polypeptides of the invention contain avanadium prosthetic group, and accordingly they are vanadiumhaloperoxidases. In another preferred embodiment, the polypeptides ofthe invention are chloroperoxidases.

In a fifth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ IDNO:2. The immunochemical properties are determined by immunologicalcross-reaction identity tests by the well-known Ouchterlony doubleimmunodiffusion procedure. Specifically, an antiserum containingpolyclonal antibodies which are immunoreactive or bind to epitopes ofthe polypeptide having the amino acid sequence of SEQ ID NO:2 areprepared by immunizing rabbits (or other rodents) according to theprocedure described by Harboe and Ingild, In N. H. Axelsen, J. Krøll,and B. Weeks, editors, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone andThorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pages 27-31). A polypeptide havingimmunochemical identity is a polypeptide, which reacts with theantiserum in an identical fashion such as total fusion of precipitates,identical precipitate morphology, and/or identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of immunochemical identity is described by Axelsen, Bock,and Krøll, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, A Manualof Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 10. A polypeptide having partialimmunochemical identity is a polypeptide, which reacts with theantiserum in a partially identical fashion such as partial fusion ofprecipitates, partially identical precipitate morphology, and/orpartially identical electrophoretic mobility using a specificimmunochemical technique. A further explanation of partialimmunochemical identity is described by Bock and Axelsen, In N. H.Axelsen, J. Krøll, and B. Weeks, editors, A Manual of Quantitativelmmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter11.

The antibody may also be a monoclonal antibody. Monoclonal antibodiesmay be prepared and used, e.g., according to the methods of E. Harlowand D. Lane, editors, 1988, Antibodies, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 60%, even more preferably atleast 80%, even more preferably at least 90%, and most preferably atleast 100% of the haloperoxidase activity of the polypeptide of SEQ IDNO:2.

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may be a fungal polypeptide, andmore preferably a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ormore preferably a filamentous fungal polypeptide such as an Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide.

In a preferred embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

In another preferred embodiment, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium: sambucinum, Fusariumsarcochroum, Fusarium sporotrichioides, Fusarnum sulphureum, Fusariumtorulosum, Fusarium trichothecioides, Fusarium venenatum, Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderna harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

In a preferred embodiment, the polypeptide is a Geniculosporium sp.polypeptide, more preferably a Geniculosporium sp. haloperoxidase, andmost preferably a Geniculosporium sp. haloperoxidase encoded by thenucleic acid sequence contained in the plasmid contained in E. coliDH10B, deposited as DSM 13442, e.g., the polypeptide with the amino acidsequence of SEQ ID NO:2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other non-haloperoxidase polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequences,which encode a polypeptide of the present invention. In a preferredembodiment, the nucleic acid sequence is set forth in SEQ ID NO:1. Inanother more preferred embodiment, the nucleic acid sequence is thesequence contained in the pUC19 derived plasmid contained in Escherichiacoli DH10B, deposited as DSM 13442. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO:2, which differ from SEQ ID NO:1 byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO:1 which encode fragments of SEQ IDNO:2 that have haloperoxidase activity.

A subsequence of SEQ ID NO:1 is a nucleic acid sequence encompassed bySEQ ID NO:1 except that one or more nucleotides from the 5′ and/or 3′end have been deleted.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the polypeptide coding sequence ofSEQ ID NO:1, in which the mutant nucleic acid sequence encodes apolypeptide which consists of the amino acid sequence of SEQ ID NO:2.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Geniculosporium, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

The present invention also relates to nucleic acid sequences which havea degree of homology to the polypeptide coding sequence of SEQ ID NO:1of at least about 80%, preferably about 90%, more preferably about 95%,and most preferably about 97% homology, which encode an activepolypeptide. For purposes of the present invention, the degree ofhomology between two nucleic acid sequences is determined by using GAPversion 8 from the GCG package with standard penalties for DNA: GAPweight 5.00, length weight 0.300, Matrix described in Gribskov andBurgess, Nucl. Acids Res. 14(16); 6745-6763 (1986).

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO:1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleic acid sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor haloperoxidase activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labelling (see, e.g., de Voset al., 1992, Science 255: 306-312; Smith et al., 1992, Journal ofMolecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize underlow stringency conditions, preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with the nucleic acid sequence of SEQ ID NO:1 or itscomplementary strand; or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under low, medium, medium-high, high,or very high stringency conditions with (i) the nucleotide sequence ofSEQ ID NO:1, (ii) a subsequence of (i), or (iii) a complementary strandof (i), (ii) or (iii); and (b) isolating the nucleic acid sequence. Thesubsequence is preferably a sequence of at least 100 nucleotides such asa sequence, which encodes a polypeptide fragment which hashaloperoxidase activity.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe polypeptide coding sequence of SEQ ID NO:1 or a subsequence thereof,wherein the mutant nucleic acid sequence encodes a polypeptide whichconsists of the amino acid sequence of SEQ ID NO:2 or a fragment thereofwhich has haloperoxidase activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences, which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” is defined herein as anucleic acid sequence, which directly specifies the amino acid sequenceof its protein product. The boundaries of a genomic coding sequence aregenerally determined by a ribosome binding site (prokaryotes) or by theATG start codon (eukaryotes) located just upstream of the open readingframe at the 5′ end of the mRNA and a transcription terminator sequencelocated just downstream of the open reading frame at the 3′ end of themRNA. A coding sequence can include, but is not limited to, DNA, cDNA,and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acid.sequence encoding a polypeptide. The term “operably linked” is definedherein as a configuration in which a control sequence is appropriatelyplaced at a position relative to the coding sequence of the DNA sequencesuch that the control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence that is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich, exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers, which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers, whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for integration of the vector into the genome by homologousor nonhomologous recombination. Alternatively, the vector may containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans and Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9,1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are generally characterizedby a mycelial wall composed of chitin, cellulose, glucan, chitosan,mannan, and other complex polysaccharides. Vegetative growth is byhyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred embodiment, the filamentous fungal host cellis a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestis,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide. Preferably, the strain is of the genusGeniculosporium.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the polypeptide coding region of SEQ ID NO:1,wherein the mutant nucleic acid sequence encodes a polypeptide whichconsists of the amino acid sequence of SEQ ID NO:2, and (b) recoveringthe polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell, which has been transformed with a nucleic acid sequenceencoding a polypeptide having haloperoxidase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as festuca, lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers.

Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct,which comprises a nucleic acid sequence encoding a polypeptide of thepresent invention operably linked with appropriate regulatory. sequencesrequired for expression of the nucleic acid sequence in the plant orplant part of choice. Furthermore, the expression construct may comprisea selectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron, which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et at., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281, Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having haloperoxidase activity of the presentinvention under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention. Preferably, thecompositions are enriched in a polypeptide of the present invention. Inthe present context, the term “enriched” indicates that thehaloperoxidase activity of the composition has been increased, e.g.,with an enrichment factor of 1.1.

The composition may comprise a polypeptide of the invention as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase. The additionalenzyme(s) may be producible by means of a microorganism belonging to thegenus Aspergillus, preferably Aspergillus aculeatus, Aspergillusawamori, Aspergillus niger, or Aspergillus oryzae, or Trichoderma,Humicola, preferably Humicola insolens, or Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Detergent Composition

The haloperoxidase of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the haloperoxidase of the invention. The detergent additiveas well as the detergent composition may comprise one or more otherenzymes such as a protease, a lipase, a cutinase, an amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a laccase, and/or aperoxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g. of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167,170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™, Primase™, Everlase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. fromB. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (αand/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264,304, 305, 391,408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g. from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenois having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid- forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the haloperoxidase of the invention, may be addedin an amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liqour, preferably 0.05-5 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

The haloperoxidase of the invention may additionally be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference.

Uses

The present invention is also directed to methods for using thepolypeptides having haloperoxidase activity.

The present invention is further directed to methods of oxidizing ahalide ion to the corresponding hypohalous acid, comprising reacting thehalide ion and a source of hydrogen peroxide in the presence of ahaloperoxidase of the invention. The present invention also relates tomethods of halogenating a compound comprising reacting the compound, ahalide ion and a source of hydrogen peroxide in the presence of a.haloperoxidase of the invention.

The present invention also relates to methods for killing or inhibitingthe growth of microbial cells, comprising contacting the cells with ahaloperoxidase of the invention, a source of hydrogen peroxide, and asource of halide or thiocyanate in an aqueous solution.

The source of hydrogen peroxide can be hydrogen peroxide itself or ahydrogen peroxide precursor, such as, a percarbonate, perborate,peroxycarboxylic acid or a salt thereof. Furthermore, the source may bea hydrogen peroxide generating enzyme system, such as an oxidase, e.g.,a glucose oxidase, glycerol oxidase or amino acid oxidase, and itssubstrate. The hydrogen peroxide source may be added in a concentrationcorresponding to a hydrogen peroxide concentration in the range of fromabout 0.001 to about 10 mM, preferably about 0.01 to about 1 mM.

The halide source may be a halide salt, preferably a sodium or potassiumsalt, such as sodium chloride, potassium chloride, sodium bromide,potassium bromide, sodium iodide, or potassium iodide. The thiocyanatesource may be a thiocyanate salt, preferably a sodium or potassium salt.

The concentration of the halide source will typically correspond to0.001-1000 mM, preferably in the range of from 0.005-500 mM, morepreferably in the range of from 0.01-100 mM, and most preferably in therange of from 0.05-50 mM.

The haloperoxidases may be used as preservation agents and disinfectionagents such as in water based paints and personal care products, e.g.,toothpaste, mouthwash, skin care creams and lotions, hair care and bodycare formulations, solutions for cleaning contact lenses and dentures.The haloperoxidases also may be used for cleaning surfaces and cookingutensils in food processing plants and in any area in which food isprepared or served. The haloperoxidases also may be used in enzymaticbleaching applications, e.g., pulp bleaching and stain bleaching (indetergent compositions).

The concentration of the haloperoxidase in the methods of use of thepresent invention, is preferably in the range of 0.01-50 mg/l, morepreferably in the range of 0.1-10 mg/l.

DNA Recombination (shuffling)

The nucleotide sequence of SEQ ID NO:1 may be used in a DNArecombination (or shuffling) process. The new polynucleotide sequencesobtained in such a process may encode new polypeptides havinghaloperoxidase activity with improved properties, such as improvedstability (storage stability, thermostability), improved specificactivity, improved pH-optimum, and/or improved tolerance towardsspecific compounds.

Shuffling between two or more homologous input polynucleotides(starting-point polynucleotides) involves fragmenting thepolynucleotides and recombining the fragments, to obtain outputpolynucleotides (i.e. polynucleotides that have been subjected to ashuffling cycle) wherein a number of nucleotide fragments are exchangedin comparison to the input polynucleotides.

DNA recombination or shuffling may be a (partially) random process inwhich a library of chimeric genes is generated from two or more startinggenes. A number of known formats can be used to carry out this shufflingor recombination process.

The process may involve random fragmentation of parental DNA followed byreassembly by PCR to new full-length genes, e.g. as presented in U.S.Pat. No. 5,605,793, U.S. Pat. No. 5,811,238, U.S. Pat. No. 5,830,721,U.S. Pat. No. 6,117,679. In-vitro recombination of genes may be carriedout, e.g. as described in U.S. Pat. No. 6,159,687, WO98/41623, U.S. Pat.No. 6,159,688, U.S. Pat. No. 5,965,408, U.S. Pat. No. 6,153,510. Therecombination process may take place in vivo in a living cell, e.g. asdescribed in WO 97/07205 and WO 98/28416.

The parental DNA may be fragmented by DNA'se I treatment or byrestriction endonuclease digests as descriobed by Kikuchi et al (2000a,Gene 236:159-167). Shuffling of two parents may be done by shufflingsingle stranded parental DNA of the two parents as described in Kikuchiet al (2000b, Gene 243:133-137).

A particular method of shuffling is to follow the methods described inCrameri et al, 1998, Nature, 391: 288-291 and Ness et al. NatureBiotechnology 17: 893-896. Another format would be the methods describedin U.S. Pat. No. 6,159,687: Examples 1 and 2.

The present invention is further described by the following examples,which should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Haloperoxidase Assays

Microtiter assays are performed by mixing 100 μl of haloperoxidasesample (about 0.2 μg/ml) and 100 μl assay buffer (0.3 M sodiumphosphate; pH 7; 1.25 mM Na₃VO₄; 50 mM KBr; 0.008% phenol red).Reactions were initiated by adding 10 μl of 0.3% H₂O₂, and theabsorption at 595 nm was measured spectrophotometrically as a functionof time in a Molecular Devices Kinetic Microplate reader.

Assays using monochlorodimedone (Sigma M4632, ε=20000 M⁻¹cm⁻¹ at 290 nm)as a substrate are performed as described below. The decrease inabsorption at 290 nm is measured as a function of time. Assays areperformed in 0.1 M sodium phosphate or 0.1 M sodium acetate, 50 μMmonochlorodimedone, 10 mM KBr/KCl, and 1 mM H₂O₂ using a haloperoxidaseconcentration of about 1 μg/ml. One HU is defined as 1 micromol ofmonochlorodimedone chlorinated or brominated per minute at pH 5 and 30°C.

Example 1 Transformation and Fermentation of Aspergillus oryzae

Protoplast preparation and transformation in Aspergillus oryzae of thenucleic acid sequence encoding the Geniculosporium sp. haloperoxidasecontained in the plasmid contained in E. coli DH10B, deposited as DSM13442, was done essentially as described by Christensen et al. (1988),Biotechnology, 6:1419-1422. Transformants were plated on AMDS agarplates selecting for the ability to grow on acetamide as sole nitrogensource.

A. oryzae transformants were spore purified twice and inoculated into100 μl YP growth medium supplemented with maltose (3%), 1 mM Na₃VO₄, and0.4% Urea. Cultures were grown at 34° C. for 5 days after which theywere assayed for haloperoxidase activity. The best haloperoxidaseproducer from the transformation was inoculated into 8×125 ml baffledflasks containing YP growth medium with maltose (3%), 1 mM Na₃VO₄, and0.4% Urea and grown for 7 days at 34° C., with shaking at 200 rpm.

Example 2 Purification of Geniculosporium sp. Haloperoxidase

Glucanex™ (available from Novozymes A/S) treated fermentation broth wascentrifuged and the supernatant was filtered through a Seitz EKS filterplate (Seitz-Filter-Werke GmbH, Germany) and concentrated and washed byultrafiltration on a Filtron Minisette™ system (Filtron TechnologyCorporation, Massachusetts, USA) with an Omega type membrane (Mw cut-offof 10 kDa) to a final volume of 300 ml and a conductance less than 2 mS.

The crude enzyme preparation was filtered on a glass filter, added 6 mlof a 10 mM sodium orthovanadate, and applied onto an anion exchangecolumn (Pharmacia 26/10 with Q-Sepharose High Performance) equilibratedwith 50 mM Tris/HCl pH 7. The column was washed with equilibrationbuffer and eluted with a 0-1M sodium chloride gradient (over 10 columnvolumes) in the same buffer using a flow of 10 ml/minute. Fractions of10 ml were collected and tested for haloperoxidase activity.

Fractions showing haloperoxidase activity were pooled, concentrated byultrafiltration on an Amicon cell (membrane Mw cut-off of 10 kDa) to afinal volume of 6-7 ml. Two times 2 ml of the concentrated sample wasapplied onto a gel filtration column (Pharmacia HiLoad 16/60, Su-perdex200 High Performance) equilibrated with 50 mM sodium acetate, 100 mMNaCl pH 5.5. The column was eluted with a flow of 1 ml/minute andfractions of 1 ml were collected. Fractions from both runs showing HPOactivity were pooled giving 22 ml with A280=10.766. The pooled fractionscontained rather pure haloperoxidase showing only one band on SDS-PAGEwith a Mr close to 65 kDa.

Example 3 Thermal Stability of Recombinant Geniculosporium sp.Haloperoxidase

The thermal stability of Geniculosporium sp. haloperoxidase wasdetermined by the following procedure:

The enzyme was diluted to an absorbance at 280 nm of approx. 0.1 in a0.3 M Tris/HCl buffer pH 7. 0.5 ml portions of the dilution wasincubated at 30, 40, 50, 60, 70, and 80° C., respectively, for 15minutes and then placed on ice water. A reference sample of the dilutedenzyme was kept at 4° C. Activity of the samples was measured accordingto the phenol red assay in microwell plate using bromide as substrate,and residual activity was calculated relatively to the reference (storedat 4° C.).

Conclusion: The residual activity of Geniculosporium sp. haloperoxidaseis at least 60% after 15 minutes incubation at 70° C.

TABLE 1 Temperature Residual activity (° C.) (%) 4 100 30 103.3 40 100.250 92.8 60 84.5 70 61.9 80 14.6

Example 4 Antibacterial Activity of GeniculosPorium sp. HaloperoxidaseAgainst Escherichia coli

The antibacterial activity of the Geniculosporium sp. haloperoxidase,available from Novozymes A/S, DK-2880 Bagsvaerd, Denmark, was testedwith bromide as enhancing agent.

The antibacterial activity of haloperoxidase was tested in HEPES-buffer(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid])(pH 7.0)against Escherichia coli DSM 1576 with potassium bromide as electrondonor, and hydrogen peroxide was added as electron acceptor. The cells(approximately 10⁶ CFU/ml) were incubated with enzyme for 15 min at 40°C.

The bactericidal activity was determined by incubation in a MalthusFlexi M2060 instrument (available from Malthus Instruments Limited,England). The detection times measured by the Malthus instrument wereconverted to CFU/ml (colony forming units pr. ml.) by a calibrationcurve. Direct measurements were used when enumerating total survivalcells. By the direct measurements, the cell metabolism was determined byconsuctance measurements in the growth substrate. When the conductancechange is measurable by the Malthus instrument, a detection time (dt)will be recorded. The dt's were converted to colony counts by use of acalibration curve relating CFU/ml to dt.

Results

TABLE 2 KBr Enzyme H₂O₂ Bactericidal activity (mM) (mg/L) (mM) (logCFU/ml) (doublets) 0 0 0 −0.1/0.1  8 0 0 0.3/0.4 0 0 1 0.2/0.8 8 0 10.6/0.5 0 1 0 0.4/0.4 8 1 1 6.4*/6.4* *corresponds to a total kill ofthe test organism

Bactericidal activity is shown in the table as log₁₀ reduction in thenumber of living cells (colony forming units), thus a bactericidalactivity of 6 correspond to a kill of 10⁶ CFU/ml. A significantbactericidal activity was obtained with the Geniculosporium sp.haloperoxidase, and no significant bactericidal activity was obtainedwith any of the controls.

DEPOSIT OF BIOLOGICAL MATERIAL

An E. coli DH10B clone containing a haloperoxidase gene fromGeniculosporium sp. (SEQ ID NO:1) inserted into a pUC19 derived plasmidhas been deposited under the terms of the Budapest Treaty with theDeutsche Sammlung von Mikroorganismen. und Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Braunschweig, Germany, and given thefollowing accession number:

Deposit Accession Number Date of Deposit NN049533 DSM 13442 2000-Apr-12

The deposit was made by Novo Nordisk A/S and was later assigned toNovozymes A/S. The strain has been deposited under conditions thatassure that access to the culture will be available during the pendencyof this patent application to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and35 U.S.C. §122. The deposit represents a substantially pure culture ofthe deposited strain. The deposit is available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

What is claimed is:
 1. An isolated polypeptide having haloperoxidaseactivity, selected from the group consisting of: a) a polypeptide havingan amino acid sequence which has at least 80% homology with the aminoacid sequence of SEQ ID NO:2; b) a polypeptide which is encoded by anucleic acid sequence which hybridizes under medium stringencyconditions with (i) the nucleotide sequence of SEQ ID NO:1, (ii) asubsequence of (i) of at least 100 contiguous nucleotides, or (iii) afull complementary strand of (i) or (ii).
 2. The polypeptide of claim 1,having an amino acid sequence which has at least 95% homology with theamino acid sequence of SEQ ID NO:2.
 3. The polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:2.
 4. The polypeptide ofclaim 1, consisting of the amino acid sequence of SEQ ID NO:2 or afragment thereof, having haloperoxidase activity.
 5. The polypeptide ofclaim 4, consisting of the amino acid sequence of SEQ ID NO:2.
 6. Thepolypeptide of claim 1, which is encoded by the nucleic acid sequencecontained in the plasmid contained in E. coli DH10B, deposited as DSM13442.
 7. A method for producing the polypeptide of claim 1, comprising(a) cultivating a strain to produce a supernatant comprising thepolypeptide; and (b) recovering the polypeptide.
 8. A method foroxidizing a halide ion comprising reacting the halide ion and a sourceof hydrogen peroxide in the presence of the polypeptide of claim
 1. 9. Amethod of halogenating a compound, comprising reacting the compound, ahalide ion, and a source of hydrogen peroxide in the presence of thepolypeptide of claim
 1. 10. A method for killing microbial cells orinhibiting growth of microbial cells, comprising contacting the cellswith the polypeptide of claim 1, a source of hydrogen peroxide, and asource of halide or thiocyanate in an aqueous solution.
 11. A detergentcomposition, comprising a surfactant and the polypeptide of claim 1.